Project Summary: Perturbation of Polycomb-mediated gene repression by histone H3 mutations Recurrent missense mutations in genes encoding histone H3 that substitute lysine to methionine (H3 ?K-to-M? mutations) were recently identified in diffuse intrinsic pontine glioma (H3K27M) and a subset of head and neck squamous cell carcinomas and undifferentiated pediatric sarcomas (H3K36M) that together are characterized by poor prognosis and have few effective treatment options. Biochemical and structural data indicate that H3K27M and H3K36M dominantly inhibit the specific histone methyltransferases (HMTs) for each lysine in trans. However, how these histone mutants promote malignant transformation is largely unknown, with initial work implicating aberrant Polycomb-mediated gene repression in cells expressing either mutation. My preliminary evidence suggests that crosstalk between histone methylation and DNA methylation is responsible for redistribution of H3K27me3-binding Polycomb Repressive Complex 1 (PRC1) in H3K36M mesenchymal progenitor cells that promotes a neoplastic undifferentiated state through derepression of polycomb target genes. In Aim 1, I plan to (a) test the hypothesis that loss of the DNA methyltransferases DNMT3a and DNMT3b recapitulates the chromatin landscape changes elicited by H3K36M, and (b) determine if recruitment of DNMT3a/b to chromatin is impacted downstream of HMT inhibition by H3K36M. Expression of H3K27M similarly derepresses polycomb target genes, however my preliminary data suggests that it is not phenotypically equivalent to chemical inhibition of the H3K27 methyltransferase PRC2, possibly due to the persistence of H3K27me3 islands at select genomic loci that have been implicated in DIPG tumor maintenance. To better understand the formation and function of these persistent islands, in Aim 2 I seek to (a) identify genes that maintain Polycomb-mediated repression in H3K27M cells, and (b) determine if the genomic position of H3K27M influences where H3K27me3 islands form genome-wide. Through these studies, I expect to gain insight into the chromatin landscape changes induced by H3 ?K-to-M? mutations that perturb Polycomb-mediated gene repression to drive oncogenesis, which will provide a basis for developing new therapeutic strategies.